This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A systematic study of nanoflow LC-MS/MS conditions (column length, gradient length) was conducted to evaluate the relationship between chromatographic performance and peptide/protein identification in a highly complex mixture. A specialized software platform was developed and used to determine fundamental performance metrics including peak capacity in relation to peptide and protein ID from data dependent acquisition. The optimum analytical strategy appears to involve the use of "short gradient-short column" (for throughput) or "long gradient-long column" (for ID quantity) approaches.